The GTP-regulatory unit (N) of the turkey erythrocyte adenylate cyclase system contains tightly bound GDP which affects both the binding of isoproterenol to the receptor and prevents activation by the normal activator, GTP. Removal of bound GDP results in restoration of the enzyme system to a tighter binding configuration of the beta adrenergic receptor and to negative heterotropic effects of hormone and GTP on the receptor-nucleotide regulatory protein complex. A separate series of studies with the human erythrocyte demonstrated that it contains an N unit that is not complexed with catalytic or receptor units. By a complementation assay with S 49 lymphoma cell variant (designated N-) that contain a C but not an N unit, it was found that the N unit in the human erythrocyte mediates the actions of guanine nucleotides, fluoride ion, and cholera toxin. Some properties of the N unit were evaluated both in its free and complexed form with the catalytic unit.